The proposed work concerns the growth cones of vertebrate CNS neurons, focusing on the developmental transition from growth cone to synaptic arbor. It is designed to learn more about signals that may regulate the activity of growth cones and to assess possible synaptogenic roles of their filopodia. The experimental strategy comprises continued use of video-enhanced contrast differential interference contrast microscopy (VEC-DICM) in tandem with the high voltage electron microscope (HVEM). Using this state-of-the-art microscopy, one first can record the real-time activity of living neurons at greater than 10,000X magnification, and then, after fixation, examine neurites, growth cones, filopodia, and synapses as whole mounts to obtain ultrastructural images that have remarkable perspective and detail. Circumventing time-lapse photography as well as traditional embedding and sectioning, this approach makes it possible to obtain new images of cellular activity and organization and to obtain these data quickly. The work will be done primarily with avian retina neurons in culture. Culture models for CNS differentiation provide ease of observation and experimentation. The retina, with its abundance of local circuit interactions and well-defined cell types, is well-suited to cell culture and is widely-used in studies of development and synaptogenesis. Four hypotheses provide a framework for the current experimental aims. While speculative, they are supported by some intriguing observations made during past project period: To test the hypothesis that formation of microtubule loops may terminate neurite extension; To test the hypothesis that neurohumoral agents may influence the activity and structure of vertebrate CNS growth cones; To test the hypothesis that filopodia may be structural precursors of synapses; To test the hypothesis that formation of cleft-spanning filaments may stabilize nascent synapses. An investigation of these four hypotheses should provide deeper understanding of how neurons become polarized for synaptic function as well as give insight into how synapses may be maintained, modified or regenerated later in life.